Engineering a cyanobacterial cell factory for production of lactic acid

Abstract

Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO(2) has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an l-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to l-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

Fig 1

Heterologous gene insertion in the cyanobacterial genome at a neutral docking site. (A) Schematic presentation of the plasmid construction scheme for subsequent integration of two heterologous genes (B. subtilis ldh and P. aeruginosa sth) into the neutral docking site, slr0168, of Synechocystis sp. PCC6803, resulting in a disruption of the open reading frame (ORF) of slr0168. (B) Verification by PCR of the insertion of the genes into the Synechocystis genome and proof of complete segregation of the ensuing two types of genomes in single colonies of the organism with primers flanking the insertion site. Panels C (ldh) and D (sth) show the verification of the insertions with specific primers for the two genes of interest. PCR on plasmids used for transformation served as positive controls. NC, negative control; M, marker.

Fig 2

Growth and extracellular lactate formation in engineered strains of Synechocystis sp. PCC6803, carrying a heterologous lactate dehydrogenase gene (ldh), cultured in continuous light (A) or in a light/dark cycle (B). Cells were grown in BG-11 medium supplemented with 50 mM NaHCO₃. Filled symbols represent the OD₇₃₀s; open symbols represent the lactate concentrations. SAA013, the strain with integrated E. coli ldh, is represented by diamonds (assaying for d-lactate); SAA015, the strain with integrated B. subtilis ldh, is represented by squares (assaying for l-lactate). Values are the averages for biological replicates; error bars indicate the standard deviations (n = 3); if error bars are not visible, they are smaller than the data point symbol. Cultures were grown under continuous white-light illumination (30 μE/m²/s; A) or under a light/dark cycle of 16 h of light (30 μE/m²/s) and 8 h of darkness (B). Gray-shaded areas represent the dark periods. SAA013 did not show l-lactate production, and SAA015 did not show d-lactate (data not shown).

Fig 3

BG-11-containing agar plate showing growth of the strain with integrated P. aeruginosa sth (SAA016) interspersed with cells that reverted to a wild-type growth phenotype. SAA016 was grown for 2 weeks on a BG-11-containing agar plate, supplemented with kanamycin. SAA016 formed colonies or a lawn of slowly growing cells (black arrows), which are interspersed with cells that reverted to a faster-growth phenotype, i.e., resembling the wild type (white arrows).

Fig 4

Growth and extracellular l-lactate formation in wild-type Synechocystis and in two of the engineered derivatives. Cells were grown in plain BG-11 medium under white-light illumination (30 μE/m²/s). Filled symbols represent the OD₇₃₀; open symbols represent the lactate concentration. The wild type is represented by circles; SAA015, the strain with integrated B. subtilis ldh, is represented by squares, and SAA017, the strain with integrated B. subtilis ldh and P. aeruginosa sth, is represented by triangles. Values are the averages for biological replicates; error bars indicate the standard deviations (n = 3); if an error bar is not visible, it is smaller than the respective data point symbol.

Fig 5

Working model of the proposed mechanism for the suppression of the genetic reversion occurring in SAA016, as opposed to SAA017. A hypothesized schematic overview of the nicotinamide-adenine dinucleotide metabolism in Synechocystis is given. (A) The wild-type organism, equilibrating its redox balance. (B) SAA016, the strain with integrated P. aeruginosa sth, showing growth reduction caused by STH activity. (C) SAA017, the strain with the additionally integrated B. subtilis ldh gene in which the genetic reversions are prevented, presumably due to the relaxation of the redox imbalance by the added NADH-consuming LDH. CO₂ fixation occurs in the Calvin-Benson cycle; LDPS, light-dependent photosynthetic electron transport; E, native energy-coupled transhydrogenase activity; STH, soluble transhydrogenase activity; LDH, lactate dehydrogenase activity.